摘要
本实验拟通过观察重复性低氧对经典型蛋白激酶C(cPKC)膜转位水平(激活程度)的影响,初步探讨cPKC特定亚型在脑低氧预适应发生过程中的作用。按我室已建立的小鼠低氧预适应模型方法,制备重复性低氧1-4次的小鼠(H1-H4)。应用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白印迹(Western bolt)等生化技术,并结合Gel Doc凝胶成像系统,半定量检测小鼠海马和大脑皮层组织内cPKCα和γ的膜转位水平。实验结果表明,随低氧次数(H1-H4)的增加,小鼠海马组织内cPKCγ的膜转位水平明显增高,且在H2、H3和H4组的变化具有统计学显著意义(P<0.05,n=6);同样,大脑皮层内cPKCγ膜转位水平也随低氧次数的增加(H1-H4)而明显增高,且在H2、H3和H4组的变化具有统计学显著意义(P<0.05,n=6):而cPKCα亚型无论在大脑皮层还是在海马组织内的膜转位变化均无统计学意义。上述观察结果提示,cPKCγ膜转位可能在脑低氧预适应的发生发展过程中发挥着重要作用;但cPKCβ Ⅰ、β Ⅱ以及其它新奇型和非典型PKC特定亚型的变化还有待于进一步的研究和探讨。
Cerebral hypoxic preconditioning (CHP), which was induced by repetitive sub-lethal hypoxic insult, is an endogenous protection of neuron against subsequent severe hypoxic injury. Although a number of possible induction pathways have been investigated, such as neuroactive cytokines, activation of glutamate receptors, the ATP-sensitive potassium channel, nitric oxide and oxidative stress, the exact mechanism underlying CHP-induced protection remains unclear. It is interesting that all the above-mentioned mechanisms are involved in the activation of protein kinases C (PKC). Recently we reported that the level of PKCs membrane translocation was significantly increased in the brain of hypoxic preconditioned mice. In order to explore the role of conventional protein kinases C (cPKC) in the development of cerebral hypoxic preconditioning, biochemical techniques of SDS-PAGE and Western bolt were applied to observe the effects of repetitive hypoxic exposure (H1-H4) on the level of cPKCα and y membrane translocation in the cortex and hippocampus of mice. Experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals. The hypoxic preconditioned mice model was adapted with minor modification from our previous report. In brief, healthy adult BALB/C mice weighing 18-20 g of either sex were randomly divided into 5 groups: control group (H0), hypoxic control group (H1, hypoxic exposure once ), hypoxic preconditioned group (H2-H4, repetitive hypoxic exposure for 2-4 times respectively). The first sign of gasping breath was taken as the end of each hypoxic exposure, and then the mice were kept in normal control condition for a 30-min interval to recover before the following hypoxic insult. We found that the level of cPKCγ membrane translocation was increased significantly (P<0.05, n=6) with the increase of the hypoxic exposure times in both hippocampus (H0: 100% vs H1-H4: 119.2%±7.0%, 139.3%±7.4%, 134.2% ± 8.95%, 184.0%±10.8%) and cortex (H0: 100% vs H1-H4: 129.7%±13.8%, 143.3%±13.9%, 204.0%±12.1%, 229.5%±14.6%) of mice. But there were no significant changes in cPKCα membrane translocation in cortex and hippocampi of hypoxic preconditioned mice. These results suggest that cPKCγ plays an important role in the development of cerebral hypoxic preconditioning. The changes in some other forms of novel and atypical PKCs are still under investigation.
出处
《生理学报》
CAS
CSCD
北大核心
2004年第4期461-465,共5页
Acta Physiologica Sinica
基金
This work was supported by the Excellent Young Teachers Program of Ministry of Education of Chian
Scientific Developing Program of Beijing Municipal Commission of Education (200KJ080
KM200310025100)
Beijing Natural Science Foundation (7032005)
Nati